gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing

J Biosci Bioeng. 2019 Sep;128(3):373-378. doi: 10.1016/j.jbiosc.2019.02.009. Epub 2019 Apr 19.

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is one of the most powerful tools for genome engineering. However, some of the steps are laborious, reducing its usability. In this study, we have developed a simplified method, called the guide RNA-transient expression system (gRNA-TES), to deliver gRNA in yeast. In gRNA-TES, a DNA fragment containing the promoter and gRNA is prepared by two simple PCR steps and co-transformed with a DNA module into the host strain; all steps including PCR steps and yeast transformation are completed within 5-6 h in a single day, in contrast to conventional plasmid-based gRNA delivery systems, which require at least 3-4 days to construct and verify the gRNA-expressing plasmids. The performance of gRNA-TES was evaluated by the replacement of 150-kb, 200-kb, 300-kb, 400-kb, and 500-kb regions of yeast chromosome 4 with a DNA module. Increased numbers of transformants with a high frequency of expected replacement of even the 500-kb region were obtained with gRNA-TES as compared with transformation without gRNA-TES. In addition, the integrity of the replaced region was verified in 67%-100% of transformants tested by colony PCR. We believe that gRNA-TES will vastly increase the accessibility of CRISPR/Cas9 technology to biologists and biotechnologists by offering a simple, fast, and cost-effective tool to deliver gRNA in genome engineering. Furthermore, it might be applied to plant and animal systems if appropriate gene promoters are incorporated in the technology.

Keywords: CRISPR-Cas9; Genome engineering; Guide RNA; PCR-based; Yeast.

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Gene Editing / methods*
  • Gene Expression Regulation, Fungal
  • Gene Transfer Techniques*
  • Genetic Engineering / methods
  • Genome, Fungal
  • Organisms, Genetically Modified
  • Plasmids
  • Polymerase Chain Reaction / methods
  • Promoter Regions, Genetic
  • RNA, Guide / genetics*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Transformation, Genetic

Substances

  • RNA, Guide